Hereditary peripheral neuropathies (also known as hereditary motor sensory neuropathies, HMSN) are among the most common genetic diseases affecting the nervous system. The mildest form of human peripheral neuropathy, Charcot-Marie- Tooth (CMT) disease, causes progressive deterioration of both motor and sensory nerves, muscular atrophy, and chronic pain/fatigue in affected individuals. A majority of inherited peripheral myelinopathies are caused by duplication of a critical myelin gene, Peripheral Myelin Protein 22 (PMP22), which is classified as CMT1A. Therefore, one of the most straightforward avenues for treating this inherited myelinopathy is to achieve a relatively subtle (<2-fold) change in gene regulation. Recent proof-of-principle studies using candidate compounds to reduce PMP22 expression levels have shown beneficial effects in rodent models of CMT1A. However, these agents have not yet been shown to be effective in clinical trials, and therefore it is criticl to develop effective screening tools that can be used to identify compounds that can achieve a therapeutic reduction in PMP22 levels. Our recent studies of PMP22 regulation have elucidated novel regulatory elements in this gene, and the goal of this proposal is to design and functionally validate novel assays to identify small molecules that reduce PMP22 expression. We propose to utilize a novel technology involving custom zinc finger nucleases, which will allow us to embed a series of orthologous reporters in the native Pmp22 locus. The assays will create a series of complementary assays for use in both primary and secondary screening assays. A series of validation assays are proposed to determine if the reporter assays are appropriately regulated, and such assays will be adapted for high throughput screening at the NIH Chemical Genomics Center.